We previously described a calcium- and calmodulin (CaM)-dependent protein kinase in synaptosomal cytosol. This kinase catalyzes the phosphorylation of several endogenous neuronal proteins. The protein kinase and major substrates bind to CaM-Sepharose in a calcium-dependent manner, and can be eluted by addition of EGTA to the eluent. Affinity chromatography using CaM-Sepharose results in a 20-fold purification of the enzyme. However, additional procedures result in a loss of enzyme activity. Therefore, we studied the stability of this protein kinase under various conditions. It appears that there are two different mechanisms involved in loss of enzyme activity. One is calcium-dependent and results in loss of total enzyme, the other is calcium-independent and results in loss of calmodulin sensitivity.